OBJECTIVE:To clone,prokaryotic express and characterize the TEM-type ?-lactamase produced by Enterobacter cloacae clinical isolate EC002. METHODS: The drug susceptibility of Enterobacter cloacae clinical isolate EC002 was detected by agar double dilution,double disk screening and confirmatory test were employed to detect the ESBLs. The isoelectric point (pI) of enzyme was detected by isoelectric focusing electrophoresis (IEF),the genes were coded by PCR amplification enzyme,and the prokaryotic expression and phenotype of the TEM-type ?-lactamase were detected. RESULTS: Enterobacter cloacae EC002 were resistant to most of the ?-lactamases. Positive results were noted for the phenotype identification and plasmid conjugation test. IEF showed that Enterobacter cloacae EC002 produced two ?-lactamases with pI value at 8.7 and 5.4 respectively,which were confirmed to be CTX-M-22 and a new TEM-subtype ?-lactamase by DNA sequencing,and the phenotype of the expressed enzyme of the cloned strains was non-ESBLs. The TEM-type ?-lactamase was named as TEM-141 by GenBank. CONCLUSION: The TEM-141 produced by Enterobacter cloacae EC002 was a new type of plasmid-mediated broad-spectrum ?-lactamase.

OBJECTIVE To study the molecular mechanism of the drug-resistance of Gram-negative bacilli to the third generation of cephalosporins.METHODS MICs of 13 ?-lactams to the eleven Gram-negative bacilli clinical isolates were detected with the standard agar dilution technique.K-B disc confirmatory method was conducted to determine the ESBLs phenotype of the eleven isolates.The ESBLs encoding genes were analyzed by using PCR.RESULTS The eleven isolates were all resistant to the third generation of cephalosporins(MIC≥64 ?g/ml).Disk confirmatory test showed that 10 isolates produced ESBLs.The hydrolytic activity of the ESBLs from the 10 isolates to cefoperazone and cefamandole was very high.However,the hydrolytic activity of the ESBLs from the 10 isolates to ceftazidime was very low.CONCLUSIONS The enzyme activities and the genes of extended-spectrum ?-lactamases from 10 Gram-negative bacilli clinical isolates are preliminarily analyzed.These results provide the basis for further study on the molecular mechanism of the drug-resistence of Gram-negative bacilli.

BACKGROUND: Heal-all oral biofilm is a material utilized in repairing oral mucosa and soft tissues defects and characterized by degradation, easily preparation, long preserved duration, convenient transportation and good ossification, which has been widely used in dental implant as guided bone regeneration materials.OBJECTIVE: To check the clinical effective of Heal-all oral biofllm on guided bone regeneration in dental implant.METHODS: A total of 72 patients with bone defects in the implantation area were selected as subjects, who were divided into control group and experimental group at random. Bone defects around implants were repaired by guided bone regeneration technique with BME-10X medical collagen membrane and Heal-all oral biofilm respectively. X-ray and clinical examination were taken at 1 and 3 months after implantation. The amount of new.formed bone tissue was evaluated when stage Ⅱ operation was performed.RESULTS AND CONCLUSION: In stage Ⅱ operation, osseointegration was formed between implants and bone tissue in all 72 patients. The average rate of bone formation was 92% in the experimental group while 91% in the control group. All implants were successfully repaired with implant denture. Occlusal function was restored successfully with all 72 implants during the follow-up period of 3-24 months after restoration. As an alternative option of BME-10X medical collagen membrane, Heal-all oral biofilm can be used in guided bone formation clinically.

Objective To compare the efficacy of hematoma evacuation between transsylvian-transinsular approach and transcortical approach in hypertensive basal ganglia hemorrhage.Methods The patients with hypertensive basal ganglia hemorrhage who underwent hematoma evacuation via transsylviantransinsular approach and transcortical approach were enrolled retrospectively.Demographics and baseline data,as well as the outcome (the modified Rankin scale 0-3 as good outcome and ≥4 as poor outcome) and mortality at 3 months were compared in both groups.Results A total of 68 patients with hypertensive cerebral hemorrhage (40 cases via transsylvian-transinsular approach and 28 via transcortical approach) were enrolled.There were no significant differences in the demographics and baseline data between the two groups (all P＞ 0.05).The good outcome rates in the transsylvian-transinsular approach and transcortical approach at 3 months after surgery were 52.50％ (21/40) and 21.43％ (6/28),respectively.The former is significantly higher than the latter (x2 =6.642; P=0.01); the mortalities in the transsylvian-transinsular approach and transcortical approach were 2.50％ (1/40) and 21.43％ (6/28),respectively.The former is significantly lower than the latter (Fisher's exact test,P=0.017).Conclusions The clinical efficacy of hypertensive basal ganglia hemorrhage via transsylvian-transinsular approach is better than the transcortical approach.

AIM: To study the inhibitory effects of 10-23 deoxyribozyme (10-23 DRz) on Escherichia coli ?-lactamase gene expression. METHODS: According to the gene sequence of Escherichia coli ?-lactamase gene blashv-5, 10-23 DRz and antisense oligonucleotides (As-ODN) were designed and synthesized. 10-23 DRz, As-ODN or control oligonucleotides were respectively introduced into Escherichia coli by the method of electroporation. Following electroporation, bacterial viability in liquid medium contained ceftazidime was detected, bacterial ?-lactamase expression was analysed by using IEF-PAGE and the ?-lactamase band was measured with gel documentation-analyzing system. RESULTS: A_ 600 in 10-23 DRz transfected Escherichia coli was lower compared with that in As-ODN transfected Escherichia coli (P

OBJECTIVE:To study the characteristics of ceftazidime-resistant?-lactamase produced by Escherichia coli.METHODS:The types of2strains of drug fast?-lactamase produced by Escherichia coli were determined initially by K-B slip diffusion method and ampholine electrophoresis method;The plasmid was extracted by alkaline lysis and the PCR ampli?fication and sequencing were conducted;?-Lactamase was counter-extracted by saturated ammonium sulfate,filtrated by Sephadex G-75gel and purified by DE-52anion exchange chromatography;The molecular weight of which was determined by SDS-PAGE and the enzyme kinetics parameters of?-lactamas were determined by ultraviolet spectrophotometry.RE?SULTS:The2strains produced a super-broad spectrum?-lactamase(CTX-M-1V)with isoionic point at8.7and the molecular weight at29kDa,which can hydrolyze cefotaxim and aztreonam but imipenem and which was sensitive to sulbactam(IC 50 =94nmol/L)and tazobactam(IC 50 =5nmol/L).CONCLUSION:CTX-M-1V is a CTX-M type super-broad spectrum?-lactamase sensitive to suppressants.

Objective To compare the vascularization of collagen scaffolds with or without growth factors and their efficacy on cardiac function in postinfarcted rats underwent surgical ventricular restoration.Methods Collagen scaffolds were activated with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride chemistry (EDC) as control or continually covalently immobilized with vascular endothelial growth factor (VEGF) and VEGF + basic fibrohlast growth factor (bFGF) as experimental groups.Adult SD male rats underwent left anterior descending artery (LAD) ligation to generate transmural myocardial infarction(MI).Four weeks later,by echocardiography,rats with moderate scar size(25％-35％ akinetic area of freedom wall of left ventricle) were screened out,assigned into 3 groups randomly and received the surgical ventricular restocation (SVR).Then,cardiac function was measured by echocardiography at 1w,2w and 4w after patch implantation.At endpoint of study (4w after patch implantation),the rats were sacrificed and the hearts were harvested.Vascularization of patch were determined by capillary density (evidenced by vWFⅧ staining) or mature vessel density (evidenced by SMA staining) respectively.Results The general mortality of the animal model is 15％ (6/40).A significant improvement of cardiac function was observed in all animals at 1 w after patch implantation but that was better preserved in both cytokine-conjugated groups 4w later (control group vs.VEGF group,P ＜ 0.05,control group vs.VEGF + bFGF group,P ＜ 0.01).More capillaries were present in patch with growth factors (P ＜0.05),while significant functional vessel formation was observed only in VEGF + bFGF group (P ＜0.01 vs.control or VEGF group).Additionally,we identified a positive correlation between heart function and mature vessel density (P =0.0297,r2 =0.998).Conclusion The mechanical property of collagen scaffold can be effectively improved by EDC,the growth factors immobilized in scaffold were in favor of vascularization of patch,which may facilitate the preservation of cardiac function posterior to SVR.

Objective:To further improve the understanding of paroxysmal nocturnal hemoglobinuria (PNH), we retrospectively analyzed and summarized the clinical characteristics, treatment status, and survival status of patients with PNH in Zhejiang Province.Methods:This study included 289 patients with PNH who visited 20 hospitals in Zhejiang Province. Their clinical characteristics, comorbidity, laboratory test results, and medications were analyzed and summarized.Results:Among the 289 patients with PNH, 148 males and 141 females, with a median onset age of 45 (16-87) years and a peak onset age of 20-49 years (57.8% ). The median lactic dehydrogenase (LDH) level was 1 142 (604-1 925) U/L. Classified by type, 70.9% (166/234) were classical, 24.4% (57/234) were PNH/bone marrow failure (BMF), and 4.7% (11/234) were subclinical. The main clinical manifestations included fatigue or weakness (80.8%, 235/289), dizziness (73.4%, 212/289), darkened urine color (66.2%, 179/272), and jaundice (46.2%, 126/270). Common comorbidities were hemoglobinuria (58.7% ), renal dysfunction (17.6% ), and thrombosis (15.0% ). Moreover, 82.3% of the patients received glucocorticoid therapy, 70.9% required blood transfusion, 30.7% used immunosuppressive agents, 13.8% received anticoagulant therapy, and 6.3% received allogeneic hematopoietic stem cell transplantation. The 10-year overall survival (OS) rate was 84.4% (95% CI 78.0% -91.3% ) . Conclusion:Patients with PNH are more common in young and middle-aged people, with a similar incidence rate between men and women. Common clinical manifestations include fatigue, hemoglobinuria, jaundice, renal dysfunction, and recurrent thrombosis. The 10-year OS of this group is similar to reports from other centers in China.

Objective To investigate the feasibility of animal model of the reconstruction of right ventricular outflow tract in rats. Methods A total of 15 female Sprague-Dawley (SD) rats underwent right ventricular outflow tract reconstruction surgery. Before the operation, the collagen scaffolds were treated with g 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride chemistry (EDC), and seeded with human bone marrow stem cells (h-MSCs). Three days after the surgery, 3 rats were randomly sacrificed to evaluate the transmural resection of right ventricular outflow tract. One or 3 months later, other 3 rats at each timepoint were sacrificed, stained with Masson’s Trichrome to observe the degradation of scaffold. Furthermore, 4 weeks after the surgery, 4 rats were sacrificed and the hearts were sliced. Anti-human mitochondria staining was used to identify the survival of seeding cells. Results The transmural resection of right ventricular outflow tract was feasible in rats at an acceptable mortality (13.3%). After EDC treatment, the degradation rate of collagen scaffold was extended greatly. The seeding cells were detected by anti-mitochandria immunofluorescent staining in all patches 4 weeks after the operation. Conclusion Rat model of right ventricular outflow tract reconstruction could be a stable, reliable and economical screening model for engineered heart tissue research.